TransAM® C/EBP α/β

TransAM® Kits are DNA-binding ELISAs that facilitate the study of transcription factor activation in mammalian tissue and cell extracts. Assays are available for over 40 different targets (see the list at right). Each kit includes a 96-stripwell plate in which multiple copies of a specific double-stranded oligonucleotide have been immobilized. When nuclear or whole-cell extract is added, activated transcription factor of interest binds the oligonucleotide at its consensus binding site and is quantified using the included antibody, which is specific for the bound, active form of the transcription factor being studied. For complete details, click the TransAM® Method tab below.

TransAM® C/EBP α/β Transcription Factor ELISA Kits

TransAM C/EBP (CCAAT-enhancer binding protein) Kits provide everything needed to study activated C/EBPα or C/EBPβ, including a positive control extract. The C/EBP α/β kit can be used with human, mouse and rat extracts. Click the C/EBP Info tab below for data and more information; kit manuals can be downloaded under the Documents tab.

C/EBP Transcription Factor Info

C/EBPs plays an important role in regulating the balance between cell growth and differentiation. The family of C/EBP (CCAAT-enhancer binding protein) basic leucine zipper transcription factors includes C/EBPα, C/EBPβ, C/EBPδ, C/EBPγ and C/EBPε. Each member exhibits similar DNA-binding specificities and contains a leucine zipper dimerization region. C/EBPs can bind DNA as homodimers and recruit coactivators to promote gene expression. C/EBPα can also heterodimerize with C/EBPβ and C/EBPγ. The TransAM C/EBP α/β Kit contains antibodies specific for the active form of C/EBPα and C/EBPβ when bound to the target DNA.

Figure 1: Specificity of C/EBPβ binding.

Increasing amounts of rat liver nuclear extract are assayed using the TransAM C/EBP α/β Kit in the presence or absence of competitor oligonucleotides containing the wild-type or a mutated form of the C/EBP consensus binding site.

The TransAM® transcription factor ELISA advantage

Historically, transcription factor studies have been conducted using gelshift, Western blot and reporter plasmid transfections, which are time-consuming, do not allow for high-throughput and provide only semi-quantitative results. TransAM assays are up to 100 times more sensitive than gelshift techniques, and can be completed in less than 5 hours. Because TransAM is an ELISA-based assay*, there is no radioactivity, and the high-throughput stripwell format enables simultaneous screening of 1-96 samples. Inconsistencies due to variable reporter plasmid transfections are eliminated, along with the need to construct stable cell lines.

How TransAM® transcription factor ELISAs work

The TransAM format is perfect for assaying transcription factor binding to a consensus-binding site. TransAM Kits contain a 96-stripwell plate to which the consensus-binding site oligo has been immobilized. Activated nuclear extract is added to each well and the transcription factor of interest binds specifically to this bound oligonucleotide. A primary antibody specific for an epitope on the bound and active form of the transcription factor is then added followed by subsequent incubation with secondary antibody and Developing Solution to provide an easily quantified, sensitive colorimetric readout (Figure 1).

Figure 1: Flow chart of the TransAM process.

Activated transcription factor in the cell extract binds to the consensus-binding site on the oligo immobilized in the well. Incubation with the supplied primary and secondary antibodies specifically quantifies the amount of activated transcription factor.

* Technology covered under EAT-filed patents and licensed to Active Motif. Use of TransAM in NFκB-related drug discovery may be covered under U.S. Patent No. 6,150,090 and require a license from Ariad Pharmaceuticals (Cambridge, MA, USA).